The enteric protozoan Entamoeba histolytica causes disease worldwide; parasite adherence and cytolytic events are crucial in pathogenesis of invasive amebiasis. Our objective is to define the biochemical and molecular basis of amebic adherence and cytolytic activities with application for development of a vaccine protective against invasive amebiasis. Parasite in vitro adherence, mediated by a 170 Kdalton galactose/N-acetyl-D-galactosamine (Gal/GalNAc) inhibitable surface lectin, initiates cytolysis of target cells. Amebic cytolytic activity is stimulated by phorbol esters requires amebic phospholipase A activity, an acid pH in endocytic vesicles- lethal increase in target cell free intracellular Ca++ ((CA++)). Candidates for a vaccine include the Gal/GalNAc lectin, cytolytic parasite proteins, and major amebic antigens. The specific aims and methods of this proposal are: (1) to determine the in vivo vaccine efficacy of purified Gal/GalNAc lectin in gerbil! models of intestinal colonization and liver abscess; (2) to describe protein kinase C (PKC) mediated regulation and stimulation of cytolytic activity by determining pborbol esterinduced PKC cytosol- membrane translocation and protein substrate phosphorylation; (3) to characterize the cytotoxicity of the purified Gal/GalNAc lectin, including lectin binding, effects on (Ca++), and uptake by the target cell; (4) to determine the mechanism of target cell death, including the role of Ca++ DNA degradation, phorpholipases, proteases, and membrane "pores"; 5) to characterize amebic proteins which participate in cytolytic activity (Cyt proteins) by mutagenesis and selection of histolytica clones deficient in cytolytic activity (Cyt-), comparison of Cyt- to Cyt+ amoebae by two-dimensional gel electrophoresis and immunoblotting to identify Cyt+ proteins altered in Cyt- mutants, partial amino acid sequencing of such proteins providing information for synthesis of oligonucleotide probes to screen E. bistolytica Cyt+ cDNA and genomic libraries, and determining function(s) of Cyt proteins by sequence analysis of full length DNA cloned from the genomic library and effects of anti-Cyt antibodies on amebic cytolytic activity; and (6) to determine the immunogenicity and vaccine efficacy of Cyt proteins and the major amebic antigens recognized by human immune sera by immunization of gerbils with purified B- galactosidase fusion proteins. This research will expand our knowledge of E. histolytica and contribute to eliminating it as a cause of human disease.